The invention relates to a process for the determination of local dye concentrations in animal and human tissues, in which light of differing wavelengths is irradiated into a subregion of the tissue, at least a part of the back-scattered light is collected, the diffuse reflectance (remission) is determined as a function of the wavelength and the concentration of dyes is determined from the spectral diffuse reflectance.
Such a process is known, for example, from the dissertation "Bestimmung von Hamoglobin--Oxygenierung und relativer Hamoglobin--Konzentration in biologischen Systemen durch Auswertung von Remissionsspektren mit Hilfe der Kubelka-Munk-Theorie" ("Determination of hemoglobin oxygenation and relative hemoglobin concentration in biological systems by evaluation of diffuse reflectance spectra by means of the Kubelka-Munk theory") by Wolfgang Dummler, Erlangen, 1988.
The term "local" concentration is to be understood here especially and exemplarily as the intracapillary region.
The term "dyes" is to be understood as dyes intrinsic to tissues (pigments), especially hemoglobin, but also cytochromes and supplied dyes, for which the elution kinetics is then investigated.
"Light of differing wavelengths" is generally the mixed light of a lamp (e.g., a xenon high-pressure lamp), but can also be, for example, the light of a tunable laser light source. The light is usually spectrally decomposed only after the diffuse reflection, and the intensity is evaluated as a function of the wavelength, the spectrally differing initial intensities being taken into consideration computationally.
The term "subregion" is to be understood as a region with relatively small surface area, typically in the range of 50-100 .mu.m diameter. The depth extent in the tissue depends on numerous factors and is on the order of 150 .mu.m (falloff to 1/e. As is described further below, however, the tissue volume from which the diffuse reflectance is obtained is both tissue-specific and equipment-specific and also depends on the hemoglobin concentration.
As is described in detail in the cited dissertation by Dummler, the absolute measurement of the hemoglobin concentration, for example, is affected by considerable difficulties. Therefore, the invention creates a process and a device which makes it possible to determine substantially more exactly the dye concentration and other scattering factors in the tissue, especially the hemoglobin absolute concentration.
This is achieved according to the invention in that, in one step, radiation from a first wavelength region in which the influence of the hemoglobin on the diffuse reflectance is small is irradiated and the diffuse reflectance in this wavelength region is determined, that in a separate step light from a second wavelength region in which the diffuse reflectance is dominated by the influence of the hemoglobin is irradiated into the same subregion of the tissue and the diffuse reflectance in this wavelength region is determined, that from the diffuse reflectance in the first wavelength region and at least one tissue-type-specific standard basic diffuse reflectance curve obtained in advance for both wavelength regions a tissue-person-specific standard basic diffuse reflectance curve for the second wavelength region is determined, and that from the determined tissue-person-specific standard basic diffuse reflectance curve and the measured diffuse reflectance in the second wavelength region a value for the hemoglobin concentration is obtained.
The classification of the steps, e.g., as is to be found from the numbering in the claims, is done systematically. Digits after a colon are to signify alternatives of the step indicated in the first digit; following digits without a colon are substeps of a main step. The light measurements in the two wavelength regions I and II, 1.1. and 1.2., are systematically different (sub)steps but in practice occur simultaneously, the order being of no significance. The basic measurements (0. steps) generally occur before ("in advance of") the actual measurements, but in principle can also be performed subsequently, since the actual measurement values can also be stored.
The term "basic diffuse reflectance" is understood here as the diffuse reflectance of the hemoglobin-free tissue, as can be found for example with a hemoglobin-free perfusion of the tissue.
The term "tissue-type-specific" is understood as the special features which result from the special nature of the tissue (e.g., rat liver or human skin). The term "tissue-person-specific" designates values and curves in which the actual measurement of at least one of the two diffuse reflectance curves is already incorporated, even if that be only through the influence of the measurement on the choice from a family of curves determined in advance.
The solution according to the invention has the specific advantage that, owing to the fact that in the first wavelength region where the hemoglobin has a window the basic diffuse reflectance is recognizable in comparatively unperturbed form, the influence of the basic diffuse reflectance in the second wavelength region where it is generally completely covered over by the hemoglobin influence can also be more exactly estimated and correspondingly eliminated. The more exact value thus obtained can be further refined in further process steps.
Preferably, a family of tissue-type-specific standard basic diffuse reflectance curves is obtained in advance from tissue samples of the same tissue type, and the measured diffuse reflectance curve in the first wavelength region is assigned to the closest matching branch in the first wavelength region from the family of standard basic diffuse reflectance curves, and the associated branch of this standard basic diffuse reflectance curve in the second wavelength region is selected as the tissue-person-specific standard basic diffuse reflectance curve.
Standard basic diffuse reflectance curves are understood as basic diffuse reflectance curves that were measured and stored "in advance" from a large number of tissues of the tissue type to be measured, e.g., by means of hemoglobin-free perfusion. In this embodiment of the invention, curves are determined which as a family of curves cover a large range of diffuse reflectances at one wavelength without the individual curves intersecting. From the family the curve is then selected which comes closest to the measured curve in the first wavelength region, and the other branch of this selected (tissue-type-specific) standard basic diffuse reflectance curve in the second wavelength region becomes through this selection the tissue-person-specific (standard) basic diffuse reflectance curve there. This has the special advantage that after such a family of curves is prepared it becomes possible in a simple manner to infer the (in itself unknown) behavior in the second wavelength region of the curve measured in the first wavelength region.
In an especially preferred manner, the assignment of a standard basic diffuse reflectance curve from the family of standard basic diffuse reflectance curves obtained in advance in the first wavelength region to the measured diffuse reflectance curve is accomplished in that the standard basic diffuse reflectance curve with the value at a predetermined isosbestic wavelength in the first wavelength region which is equal to or closest to the measured diffuse reflectance value at that isosbestic wavelength is selected, and the value of the selected standard basic diffuse reflectance curve at a predetermined isosbestic wavelength in the second wavelength region is used as the value for the determination of the hemoglobin concentration.
The values at the isosbestic wavelengths are taken because no additional error occurs there due to the (likewise still unknown) oxygenation of the hemoglobin. But these values also suffice for the stated purpose because the diffuse reflectance value from the diffuse reflectance curve at an isosbestic wavelength, corrected by the basic diffuse reflectance, already suffices to determine the concentration from the diffuse reflectance value using a suitably calibrated device.
In an alternatively preferred manner, an averaged tissue-type-specific standard basic diffuse reflectance curve is obtained in advance from tissue samples of the same tissue type, and the measured diffuse reflectance curve in the first wavelength region is compared by ratio to the averaged standard basic diffuse reflectance curve, and, from the ratio obtained and the part of the tissue-type-specific averaged standard basic diffuse reflectance curve in the second wavelength region, a tissue-person-specific standard basic diffuse reflectance curve in the second wavelength region is obtained.
Thus, in distinction to the previous alternative, now one typical curve is determined from a plurality of advance measurements of the basic diffuse reflectance of the tissue (e.g., by hemoglobin-free perfusion) over both wavelength regions, which for that reason is also designated as the "averaged" standard basic diffuse reflectance curve (although the curves of the family of curves may in turn also have resulted from averagings).
"Comparison by ratio" is to be understood in any case not only as the formation of a mathematical ratio, but rather numerous methods are conceivable by which from the deviations of the behavior of the measured diffuse reflectance curve in the first wavelength region from the behavior of the averaged standard diffuse reflectance curve in the first wavelength region via the behavior of the averaged standard diffuse reflectance curve in the second wavelength region it is possible to infer the imaginary continuation of the measured curve (considered as the basic diffuse reflectance curve in zeroth approximation) as the tissue-person-specific basic diffuse reflectance curve in the second wavelength region.
In an especially preferred manner, the comparison by ratio of the averaged standard basic diffuse reflectance curve in the first wavelength region to the measured diffuse reflectance curve (in the first wavelength region) is accomplished in that the value of the averaged standard basic diffuse reflectance curve in the first wavelength region at a predetermined isosbestic wavelength in the first wavelength region is compared by ratio to the measured value of diffuse reflectance at that isosbestic wavelength, and by means of the obtained ratio the value of the averaged standard basic diffuse reflectance curve at a predetermined isosbestic wavelength in the second wavelength region is used to obtain a value of diffuse reflectance at that isosbestic wavelength, which is used as the value for the determination of the hemoglobin concentration.
The advantage of the use of the values at isosbestic wavelengths was already explained. The diffuse reflectance value, which allows the hemoglobin concentration to be inferred, is generally determined by subtracting the value obtained at the isosbestic wavelength in the second wavelength region from the measured value of diffuse reflectance at that wavelength.
In a preferred manner, in a continuation of the process, the measured curve in the first wavelength region is corrected by means of the value obtained for the hemoglobin concentration, whereby a second, improved approximation is obtained for the tissue-person-specific basic diffuse reflectance in the first wavelength region.
In the above process, the measured diffuse reflectance curve in the first wavelength region, which still contained the influence (which of course is small there) of the hemoglobin concentration, was a "zeroth approximation" (or zeroth order) of a tissue-person-specific basic diffuse reflectance curve in the first wavelength region. This zeroth approximation can now be improved by eliminating the hemoglobin concentration (which in turn is known in first approximation from the above process steps) from the curve. The further approximation thus obtained is advantageously incorporated into the above-described process steps in place of the measured diffuse reflectance curve.
Thus, in an especially preferred manner, steps 2 to 4 are performed with the improved curve instead of the measured diffuse reflectance curve, whereby a better approximation value is obtained for the hemoglobin concentration and a further improved curve is obtained as tissue-person-specific basic diffuse reflectance in the first wavelength region. In a preferred manner, the above steps 2. to 4. are repeated n, where n is a predetermined number, times, using the improved values and curves as a basis in each instance.
The region from 630 nm to 1000 nm is preferred as a wider first wavelength region.
The region from 750 nm to 850 nm is preferred as a narrower first wavelength region. Hemoglobin has a window in these regions, i.e., its influence on the diffuse reflectance is small.
The region from 500 nm to 620 nm is preferred as a wider second wavelength region.
The region from 550 nm to 570 nm is preferred as a narrower second wavelength region. In the latter two regions, the influence of hemoglobin on the basic diffuse reflectance is large.
The invention also relates to a process for the determination of the oxygenation of hemoglobin, especially using one or more of the curves from one or more of the above processes. The determination of the oxygenation of hemoglobin with high accuracy is also of special significance in the monitoring of life processes by means of spectrophotometry.
In this connection, according to the invention, a "pure" hemoglobin curve is obtained from a tissue-person-specific standard basic diffuse reflectance curve and the measured diffuse reflectance curve in the second wavelength region; a family of "pure" hemoglobin curves having been obtained in advance in the range of from 0% to 100% oxygenation by superposition of two pure standard hemoglobin curves, namely for 0% and 100% oxygenation, with different weightings; the "pure" hemoglobin curve, after normalization to 1, is compared to the likewise normalized standard hemoglobin curves of the family, the closest matching one is picked out, and its oxygenation is assumed as the value of the oxygenation for the measured curve.
In an alternatively preferred process for the determination of the oxygenation of hemoglobin, especially using one of the concentration values and especially one of the curves from one of the previously described processes, a two-dimensional family of comparison curves is prepared in advance by means of a plurality of measurements on the same tissue type with hemoglobin of differing concentrations and differing oxygenations, the comparison curves with hemoglobin concentrations in the vicinity of the determined concentration are searched throughout the entire range of oxygenation, and the best matching of the comparison curves yields an assumed value for the oxygenation and an improved value for the concentration.
In this manner, reliable values for important parameters can in turn be obtained simply and quickly with the aid of the measured diffuse reflectance and standard values known in advance.
In a preferred manner, the values for the concentration and the oxygenation obtained in the manner just described are used in the described step for obtaining an improved tissue-person-specific basic diffuse reflectance curve.
In an especially preferred manner, the measured diffuse reflectance curve from the second wavelength region is normalized to the closest matching curve from the above-mentioned two-dimensional family used to determine the oxygenation, the difference between the two curves is plotted versus wavelength and used as a measure of the distortion to determine the penetration depth of the irradiated light, i.e., of the volume V covered by the light.
It has turned out that this distortion can be used as a measure of the penetration depth. Corresponding distortion curves determined in advance are stored, the corresponding volume is assigned to them, and then the distortion curve obtained by the comparison is assigned to the closest matching curve from the stored distortion curves and thus the volume is determined.
In an especially preferred manner, the Erlanger light-guide microspectrophotometer (Erlanger Mikrolichtleiterspektrophotometer (EMPHO)) is used for the above measurements. It is described in more detail further below. The graded-density interference filter disk used in it covers preferably both the first wavelength region and also the second wavelength region. In that way, the measured diffuse reflectance curves of the first and second wavelenqth regions can be obtained in one rotation of the disk.
In a preferred manner, the light-guide microspectrophotometer has means for absolute calibration of the illuminating and detecting system. The absolute calibration is especially important because the measurements made in advance, which should be made with the same device or must be converted in a device-specific manner, must occur under defined conditions comparable to those of the actual measurement. In particular, such means are a white standard and a standard light source, which are explained below.
The invention relates also to a device for the determination of size variations of tissue particles. The observation of such variations, e.g., of the size change of mitochondria, is of special practical importance because it makes it possible, for example, to detect a cerebral edema at an early stage. This is accomplished by means of a device with a light guide radiating light into the tissue, at least two light guides at different radial distances from it [the first light guide] which receive the back-scattered light and which preferably are disposed along a line on both sides of the illuminating light guide, and an evaluation unit for each of the light guides which determines and evaluates the time variation of the back-scattered intensity relative to the other light guides.
Such an evaluation unit can be built analogously to the Erlanger light-guide microspectrophotometer. A flattening or other deformation of the back-scattering characteristic can then be recognized, which in turn allows one to infer the variation of the particle size.